慢性缺氧及高脂饮食影响兔血浆中oxLDL及Lp-PLA2表达及促动脉粥样硬化形成

摘要目的建立慢性缺氧新西兰白兔模型，观察慢性缺氧和高脂饮食对血浆中oxLDL和Lp-PLA2表达影响及促动脉粥样硬化的作用。

方法将24只健康新西兰兔随机分为对照组、高脂组、慢性缺氧组、慢性缺氧合并高脂饮食组，共4组，6只/组。

在0w、4w、8w和12w时，各组新西兰白兔取外周血，应用ELISA法检测血浆中oxLDL和Lp-PLA2含量，并于12w末取各组新西兰白兔主动脉弓和腹主动脉进行HE染色，观察血管病理改变。

统计方法经SPSS16.0中的单因素方差分析、重复测量数据分析和相关性分析。

结果1、血浆中oxLDL含量：(1) 组内比较：与0w相比，在4w、8w和12w时，除对照组外，其余3组血浆中oxLDL含量均不同程度增高（P<0.05）, 其中8w时，血浆中oxLDL含量增高最明显（P<0.05）。

(2) 组间比较：①与对照组相比，除0w 外，其余3组血浆中oxLDL含量均不同程度增高（P<0.05），其中，在4w时，与高脂组相比，慢性缺氧组血浆中oxLDL含量增高更明显（P<0.05），但在8w和12w 时，比较两组血浆中oxLDL含量，差异无统计学意义（P>0.05）；与正常对照组、高脂组和慢性缺氧组相比，在4w、8w和12w时，慢性缺氧合并高脂组血浆中oxLDL 含量增高最明显（P<0.05）。

2、血浆中Lp-PLA2含量：(1) 组内比较：与0w相比，在4w时，各组血浆中Lp-PLA2含量，差异无统计学意义（P>0.05）；与0w和4w相比，在8w和12w 时，除对照组外，其余3组血浆中Lp-PLA2含量均不同程度增高（P<0.05），其中，在12w时血浆中Lp-PLA2含量增高最明显（P<0.05）。

(2) 组间比较：与对照组相比，除0w和4w外，其余3组血浆中Lp-PLA2含量均不同程度增高（P<0.05），其中，在12w时，与高脂组相比，慢性缺氧组血浆中Lp-PLA2含量增高更明显（P<0.05）；与正常对照组、高脂组和慢性缺氧组相比，慢性缺氧合并高脂组血浆中Lp-PLA2含量在12w时增高最明显（P<0.05）。

3、各组与时间对血浆中oxLDL和Lp-PLA2含量影响：重复测量时间和实验分组对血浆中oxLDL 和Lp-PLA2水平均有显著影响（P<0.05），时间与分组之间存在交互效应（P<0.05），血浆中oxLDL 和Lp-PLA2 表达变化随时间和分组的不同而不同。

4、体重比较：各组新西兰白兔体重组内比较均不同程度增高（P<0.05）；各组间比较差异无统计学意义（P>0.05）。

5、相关性分析：通过相关性分析，我们发现体重与oxLDL和Lp-PLA2含量之间无相关性（P>0.05）。

6、组织病理学观察：在12w时，除对照组外，高脂组、慢性缺氧组、慢性缺氧组合并高脂组新西兰白兔主动脉弓及腹主动脉的血管壁上均有不同程度的动脉粥样硬化形成。

结论1、慢性缺氧和高脂饮食均能促进新西兰白兔主动脉弓及腹主动脉局部动脉粥样硬化形成。

2、慢性缺氧和高脂饮食均能促进血浆中oxLDL和Lp-PLA2含量增加，且变化随时间的不同而不同。

3、慢性缺氧与高脂饮食联合作用能使血浆中oxLDL和Lp-PLA2含量增加更明显，在促动脉粥样硬化形成和发展过程中起重要作用。

关键词：缺氧；动脉粥样硬化；氧化型低密度脂蛋白；脂蛋白相关性磷脂酶A2The Effects of Chronic Hypoxia and High Fat Diet onthe expression of OxLDL and Lp-PLA2 in Plasma ofRabbit and Promoting Atherosclerosis FormationAbstractObjective Establishment of chronic hypoxia model of New Zealand white rabbit, observation of chronic hypoxia and high fat died on the expression of oxLDL, Lp-PLA2 in plasma and promoting formation of atherosclerosis.Method Twenty-four healthy New Zealand white rabbits were randomly divided into 4 groups, Normal control group, High fat group, Chronic hypoxia and Chronic hypoxia & high adipose diet group. There are 6 rabbits in each group. At the time of the 0w, the 4th week, the 8th week and the 12th week, expression of oxLDL and Lp-PLA2 in the plasma were measured by ELISA assay, And observe the pathological changes in their aortic arch and abdominal aorta with HE staining at the 12th week. Determine whether the model is a success. All datas were analyzed by spss16.0, with repeated measure , one-way anove and bivariate.Results1. Plasma levels of oxLDL: (1) Comparison inner each group: Compared with 0th week, At 4th week, 8th week and 12th week, except the control group, plasma levels of oxLDL had certain degree influence on the other three groups(P<0.05), among which, At 8th week increased most significantly in oxLDL concentrations(P<0.05). (2) Comparison within the group: Except the 0th week, concentrations of oxLDL was significantly increased in other three groups compared with the control group (P<0.05); among which, At 4th week, the levels of oxLDL increased more significantly in the chronic hypoxia group compared with the high-fat group (P<0.05), there was no statistical significance compared with chronic hypoxia group compared with high-fat group at 8th week and 12th week(P>0.05); At 4th week, 8th week and 12th week, chronic hypoxia & high adipose diet group increased most significantly (P<0.05).2、Plasma levels of Lp-PLA2: (1) Comparison inner each group: Compared with 0th week, At 4th week, there was no statistical significance to every group (P>0.05). Compared with 0th week and 4th week, At 8th week and 12th week, except the controlgroup, plasma levels of Lp-PLA2had certain degree influence on the other three groups(P<0.05), among which, At 12th week increased most significantly in Lp-PLA2 concentrations(P<0.05). (2) Comparison within the group: Except the 0 th week and 4th week, concentrations of Lp-PLA2was significantly increased in other three groups compared with the control group (P<0.05); among which, At 12th week, the levels of Lp-PLA2 increased more significantly in the chronic hypoxia group compared with the high-fat group (P<0.05); At 12th week, chronic hypoxia & high adipose diet group increased most significantly compared with the other three groups(P<0.05).3 By repeated measurement analysis oxLDL and Lp-PLA2 levels in plasma: what the repeated measurement time and the treatment groups influence significantly on plasma levels of oxLDL and Lp-PLA2were observde. There have interaction effect between repeated measurement time and the treatment groups(P<0.05). the plasma expression of oxLDL and Lp-PLA2 were varied with different time and groups.4. Comparison of Weight: From the 0th week to the 12th week, we found that the weight of the control group, high fat group, chronic hypoxia and chronic hypoxia & high adipose diet group increased to varying degrees(P<0.05). But there is no statistic significance in the group within comparison of rabbit′s weight difference(P>0.05).5. Correlation analysis: Through correlation analysis, we found that the levels of oxLDL and Lp-PLA2 were negatively correlated with weight (P>0.05).6. Observation in pathology intimates that，except the control group，the aortas and abdominal aorta of the other groups had varying degrees of local atherosclerosis pathological changes.Conclusions1 Chronic hypoxia and high-fat diet can promote the New Zealand white rabbits aortic arch and aorta atherosclerosis formation.2. Chronic hypoxia and high-fat diet can promote the plasma expression of oxLDL and Lp-PLA2, there are varied with different time.3. There are significant increase of oxLDL and Lp-PLA2 levels and promote atherosclerosis formation in Cooperation with chronic hypoxia and high-fat diet .Key Words:hypoxia; atherosclerosis; Oxidized low density lipoprotein; lipoprotein-associatedPhospholipaseA2Directed by: Prof. LIU Guo RongApplicant for Master degree: YAN Jie （Department of Neurology) (Baotou Central Hospital, Baotou Medical College, 014040. China)目录第一章前言 (1)1.1 建立慢性缺氧动脉粥样硬化模型的提出 (1)1.2 Lp-PLA2、oxLDL、慢性缺氧和高脂饮食之间的国内外研究现状 (1)1.3 本课题研究的目的与意义 (3)第二章实验材料与方法 (3)2.1 实验材料 (3)2.1.1 实验对象及动物喂养 (3)2.1.2 实验分组 (3)2.1.3 主要试剂 (3)2.1.4 主要仪器设备及型号 (4)2.2 实验方法 (5)2.2.1 实验准备 (5)2.2.2 慢性缺氧造模方法 (5)2.2.3 动物实验技术路线流程 (6)2.2.4 血浆留取 (6)2.2.5 oxLDL和Lp-PLA2含量的测定 (6)2.2.6 标本取材 (7)2.2.7 石蜡标本的制作 (7)2.2.8 HE染色 (8)2.2.9 统计学处理 (9)第三章结果 (9)3.1 新西兰白兔生长情况 (9)3.2 各组新西兰白兔体重变化情况 (10)3.3 新西兰白兔血浆中oxLDL和Lp-PLA2含量变化 (10)3.3.1各组新西兰白兔血浆中oxLDL含量比较 (10)3.3.2 各组新西兰白兔血浆中Lp-PLA2含量比较 (12)3.4 在不同时间各组新西兰白兔体重与oxLDL和Lp-PLA2的相关性分析 (15)3.5 各组新西兰白兔主动脉弓和腹主动脉HE染色 (15)第四章讨论 (17)4.1 慢性缺氧兔模型建立及对动脉粥样硬化影响 (17)4.2 慢性缺氧和高脂饮食影响血浆中oxLDL和Lp-PLA2表达 (18)4.3 实验设计的优点及局限性 (21)第五章结论 (22)致谢 (23)参考文献 (24)文献综述 (27)作者简介 (36)学位论文独创性声明 (37)学位论文知识产权权属声明 (37)插图和附表清单1．图1 实验仪器 (5)2．图2 实验设计技术路线流程 (6)3. 图3 慢性缺氧合并高脂组新西兰白兔肺和肝叶标本 (9)4．图4 各组4次重复测量oxLDL含量的均数变化组内比较趋势图 (11)5. 图5 各组4次重复测量oxLDL含量的均数变化组间比较趋势图 (12)6．图6 各组4次重复测量Lp-PLA2含量的均数变化组内比较趋势图 (13)7. 图7 各组4次重复测量Lp-PLA2含量的均数变化组间比较趋势图 (14)8．图8 各组兔主动脉弓平片（HE染色） (16)9. 图9 各组兔腹主动脉平片（HE染色）. (16)10．表1 各组新西兰白兔体重组内和组间比较统计分析表 (10)11．表2 各组兔血浆中oxLDL含量组内比较统计分析表 (11)12．表3 各组兔血浆中oxLDL含量组间比较统计分析表 (12)13．表4 各组兔血浆中Lp-PLA2含量组内比较统计分析表 (13)14．表5 各组兔血浆中Lp-PLA2含量组间比较统计分析表 (14)14. 表6 各组兔不同时间点体重与oxLDL和Lp-PLA2的相关性分析 (15)缩略语表缩略语全称中文名称PLA2Lp-PLA2 sPLA2 cPLA2 iPLA2 LDL HDL oxLDLp38MAPK apoB-100 ROS oxPC PhospholipaseA2Lipoprotein-associated PLA 2Secretory PhospholipaseA2Cytosolic PLA2Ca2+-independent PhospholipaseA2low Density LipoproteinHigh Density LipoproteinOxidized LDLP38 Mitogen Activated Protein KinaseApoprotein B-100reactive oxygen speciesoxidized phosphatidylcholine磷脂酶A2脂蛋白相关磷脂酶A2分泌型磷脂酶A2细胞溶质型磷脂酶A2钙离子非依赖型磷脂酶A2低密度脂蛋白高密度脂蛋白氧化型低密度脂蛋白p38丝裂原活化蛋白激酶载脂蛋白B-100活性氧氧化型磷脂酰胆碱内蒙古科技大学包头医学院硕士学位论文第一章前言1.1 建立慢性缺氧动脉粥样硬化模型的提出我国脑血管发病率一直居高不下，以缺血性脑血管病较多见，约占全部脑血管病人的70%～80%，主要是由于动脉粥样硬化所致。